Development of zafirlukast analogues for improved anti-thrombotic activity through thiol isomerase inhibition

Keovilay, J. A., Howard, K. C., Taylor, K. A. ORCID: https://orcid.org/0000-0002-4599-7727, Khan, S., Wurl, S. E., Szahaj, M. K., Sage, T., Chandrika, N. T., Hou, C., Tsodikov, O. V., Gibbins, J. M. ORCID: https://orcid.org/0000-0002-0372-5352, Garneau-Tsodikova, S. and Kennedy, D. R. (2025) Development of zafirlukast analogues for improved anti-thrombotic activity through thiol isomerase inhibition. Arteriosclerosis, Thrombosis, and Vascular Biology, 45 (4). e136-e149. ISSN 1079-5642 doi: 10.1161/ATVBAHA.124.321579

Abstract/Summary

Background: Thiol isomerases play essential and non-redundant roles in platelet activation, aggregation, and thrombus formation. Thiol isomerase inhibitors have the potential to overcome the two major drawbacks of current antithrombotic therapies, as they target both arterial and venous thrombosis without enhancing bleeding risks. Recently, an FDA-approved drug, zafirlukast (ZAF), was shown to be a promising pan-thiol isomerase inhibitor. The objective of this study is to develop analogues of ZAF with optimized thiol isomerase inhibition and antithrombotic activity. Methods: 35 ZAF analogues were tested in an insulin turbidometric assay for thiol isomerase inhibition. Analogues were tested for platelet activation, aggregation, P-selectin expression, and laser-induced thrombosis in mice and compared with the parent compound. Results: Of the 35 analogues, 12 retained activity, with one, compound 21 that demonstrated a greater potency than that of ZAF, 5 had a similar potency to that of ZAF, and 6 had a weaker potency. Analogues demonstrated inhibition of platelet aggregation and P-selectin expression as compared to ZAF, consistent with their potencies. ZAF and compound 21 were shown to be reversible inhibitors of thiol isomerases, and not cytotoxic to cultured, lung, liver, and kidney cells. Finally, in an in vivo assessment of thrombus formation, compound 21 was able to significantly inhibit thrombus formation without affecting bleeding times. Conclusions: A ZAF analogue, compound 21, with properties superior to those of ZAF was synthesized, demonstrating improved inhibition of platelet activation, aggregation, and thrombus formation as compared to the parent ZAF. This approach could yield a promising clinical candidate for treatment and prophylaxis of arterial and venous thrombosis.